PROJECT SUMMARY Graft-versus-host disease (GVHD) is the most serious complication of allogeneic hematopoietic stem cell transplantation (HSCT) and occurs in approximately 50% of all HSCT recipients despite routine immune prophylaxis. GVHD is mediated by mature alloreactive donor-derived T cells, which are present in the graft at the time of transplant and ultimately cause tissue damage. During the acute phase of GVHD, which typically occurs in the first 100 days post-transplantation, inflammation is restricted to a limited number of target organs, specifically the skin, liver, and gastrointestinal (GI) tract. Damage to the GI tract from GVHD is a particularly serious event leading to significant morbidity and mortality. Proinflammatory cytokines play a critical role in the pathophysiology of intestinal GVHD by activating both the innate and adaptive arms of the immune system. While T cells are the proximate drivers of GVHD, disease induction and amplification rely on this crosstalk between innate and adaptive immune cells. However, the cellular and cytokine networks which mediate this interplay are incompletely understood. Preliminary studies using well-characterized murine models of GVHD have identified GM-CSF as a cytokine which is produced at high levels by donor-derived CD4+ T cells in the GI tract during GVHD. Moreover, disruption of GM-CSF signaling in CD4+ T cells significantly prolongs survival in murine recipients undergoing GVHD and reduces inflammatory damage to the colon, indicating that this cytokine plays an important role in GVHD biology. Based on these studies, the overall hypothesis of this proposal is that GM-CSF promotes inflammation in the GI tract via the downstream activation of innate immune populations that potentiate T cell alloreactivity. Studies in Specific Aim 1 will determine the GM-CSF responsive myeloid cell populations that mediate inflammation in the GI tract during GVHD. This aim will employ novel genetic and antibody-based approaches to identify the relevant GM-CSF- dependent innate immune populations and will determine how these cellular mediators are able to directly contribute to the proinflammatory milieu. Studies in Specific Aim 2 will define the mechanistic pathways by which GM-CSF modulates T cell alloreactivity in GVHD. These experiments will examine how GM-CSF affects the production of interleukin-23 by donor-derived antigen presenting cells (APCs), a cytokine that has previously been shown to be crucial for the induction of gastrointestinal inflammation during GVHD, determine how GM- CSF signaling in APCs affects indirect alloantigen presentation in the GI tract, and characterize how GM-CSF modulates reconstitution of the regulatory T cell compartment. The overall objective of this proposal is to provide new insights into the pathophysiology and regulation of GVHD within the GI tract that will foster the development of clinically relevant strategies to mitigate this complication in allogeneic HSCT recipients.